Project Summary The hepatitis C virus (HCV) is the leading cause of liver cancer and liver transplants in the United States. Although new treatments are very effective at curing infection, most patients worldwide will not benefit from these therapies due to cost. Furthermore, cured patients are not protected from new HCV infection. A protective vaccine would greatly augment the efforts to reduce the HCV global health impact. An immunocompetent animal model would aid development of an effective HCV vaccine, however the creation of such models has been impeded by HCV's strict species-tropism, as efficient HCV infection is restricted to only humans and chimpanzees. The experiments proposed in this application are aimed at defining and overcoming mechanisms by which HCV tropism is regulated, and are based on our hypothesis that HCV species-specific tropism is influenced, at least in part, by differences in the capacity for HCV to enter host cells and suppress innate immune responses in disparate species. We recently discovered that ferrets are able to support HCV infection in vivo, but at much lower levels than observed in humans and chimpanzees. We determined that at least two host factor interactions limit HCV infection of ferret cells. The first was that the ferret occludin (OCLN) ortholog was deficient in mediating HCV cell entry. By passing virus through cells expressing this protein, we successfully selected a mutant virus that is adapted to ferret OCLN. The second block was at the level of host immune suppression, as we have found that HCV is unable to cleave ferret MAVS. We used a phage-based protease evolution system to select a set of ferret MAVS adapted HCV protease mutations. At least one of these mutations both enhanced ferret MAVS cleavage and did not impair HCV replication. In the first aim of our propose experiments, we will to formally test our hypothesis by determining the capacity of a HCV virus that is adapted to both ferret OCLN and MAVS to infect ferrets in vivo. We will compare infection efficiency, by tracking viral RNA, infectious virus, liver enzymes, and infected cell foci size, of single and double adapted viruses to wild type and replication incompetent controls. As the single protease mutation that was tolerated by the virus was only partially enhanced ferret MAVS cleavage, in the second aim of the proposed experiments we will further adapt HCV to replicate in the presence of larger sets of ferret MAVS adapted protease mutations. Our experiments have the potential to directly provide an immunocompetent animal model for the study of HCV pathogenesis, persistence, and vaccines. Furthermore, by identifying species-specific entry, replication and innate immunity restrictions to HCV infection across a wide range of species and defining how these blocks contribute to HCV viral tropism, we can devise methods for efficient HCV infection in a range of species as well as provide insight on how host-pathogen interactions impact the life cycles and host susceptibility for a range of other viruses with similar replication and immune evasion mechanisms.